A three-dimensional (3D) organotypic microfluidic model for glioma stem cells - Vascular interactions.

Glioblastoma (GBM) is one of the deadliest forms of cancer. Despite many treatment options, prognosis of GBM remains dismal with a 5-year survival rate of 4.7%. Even then, tumors often recur after treatment. Tumor recurrence is hypothesized to be driven by glioma stem cell (GSC) populations which are highly tumorigenic, invasive, and resistant to several forms of therapy. GSCs are often concentrated around the tumor vasculature, referred to as the vascular niche, which are known to provide microenvironmental cues to maintain GSC stemness, promote invasion, and resistance to therapies. In this work, we developed a 3D organotypic microfluidic platform, integrated with hydrogel-based biomaterials, to mimic the GSC vascular niche and study the influence of endothelial cells (ECs) on patient-derived GSC behavior and identify signaling cues that mediate their invasion and phenotype. The established microvascular network enhanced GSC migration within a 3D hydrogel, promoted invasive morphology as well as maintained GSC proliferation rates and phenotype (Nestin, SOX2, CD44). Notably, we compared migration behavior to in vivo mice model and found similar invasive morphology suggesting that our microfluidic system could represent a physiologically relevant in vivo microenvironment. Moreover, we confirmed that CXCL12-CXCR4 signaling is involved in promoting GSC invasion in a 3D vascular microenvironment by utilizing a CXCR4 antagonist (AMD3100), while also demonstrating the effectiveness of the microfluidic as a drug screening assay. Our model presents a potential ex vivo platform for studying the interplay of GSCs with its surrounding microenvironment as well as development of future therapeutic strategies tailored toward disrupting key molecular pathways involved in GSC regulatory mechanisms.

Characterization of diverse homoserine lactone synthases in Escherichia coli.

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach were still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum signaling synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.